Class Of Sites Research Articles

ER regulates stress granule fission

Cell Biology A hallmark of eukaryotic cells is the ability to compartmentalize essential reactions into membrane-bound and membraneless organelles. Membrane-bound organelles form networks through transport vesicles and interorganellar contact sites. The endoplasmic reticulum (ER) has emerged as a network hub and forms physical connections with nearly every membrane-bound organelle. Lee et al. now identify another class of ER contact sites that appear to help regulate the biogenesis and fission of membraneless ribonucleoprotein (RNP) granules (see the Perspective by Kornmann and Weis). Live-cell fluorescence microscopy of human cells revealed that ER tubule dynamics are spatially and temporally coupled to the fission site of two types of RNP granules, processing bodies (P-bodies) and stress granules. Science , this issue p. [eaay7108][1]; see also p. [507][2] [1]: /lookup/doi/10.1126/science.aay7108 [2]: /lookup/doi/10.1126/science.aba3771

ADAPTASI TIGA VARIETAS PEPAYA (MERAH DELIMA, JUPE, MADU) DI LAHAN KERING DATARAN RENDAH

Papaya fruit (Carica papaya L.) is classified as having high economic value and is being developed by many farmer. A common problem is the papaya variety that farmer \s develop usually from careless seeds, so the production and quality are low. The aim of this research is to introduce superior papaya and have good quality. Three varieties papaya are used in this study, namely: Merah Delima, Jupe, and Madu.The research location is located in the dry land of Banturung, Palangka Raya City. The result that the land suitability class of the study site included S3wa,on, which is Marginal Suitability with the heaviest inhibiting factors of water availability and oxygen availability. On Growth parameters, then Merah Delima has the largest stem circumference of 10,1 cm althought it is not significantly different from other varieties 8,8 cm. While the plant height is the best Merah Delima papaya and signficantly different from Madu. Interms of Productioan Jupe has a number of Fruits (38 fruits/tree) that are significantly different from Madu, and the weight of fruit (1,2 kg/fruit) is significantly different from Merah Delima., and fruit weight per tree 34,1 kg is significantly different from the other two varieties. Base on the sweetness level, the sweetest Merah Delima reaches 9,1 oBrix significantly different from Jupe (6,9 oBrix) and Madu (6,6 oBrix). Parameters of growth, productionand quality of papaya can still be improved by improving the management of inhibiting factors.

Lipid Interactions of a Ciliary Membrane TRP Channel: Simulation and Structural Studies of Polycystin-2

SummaryPolycystin-2 (PC2) is a transient receptor potential (TRP) channel present in ciliary membranes of the kidney. PC2 shares a transmembrane fold with other TRP channels, in addition to an extracellular domain found in TRPP and TRPML channels. Using molecular dynamics (MD) simulations and cryoelectron microscopy we identify and characterize PIP2 and cholesterol interactions with PC2. PC2 is revealed to have a PIP binding site close to the equivalent vanilloid/lipid binding site in the TRPV1 channel. A 3.0-Å structure reveals a binding site for cholesterol on PC2. Cholesterol interactions with the channel at this site are characterized by MD simulations. The two classes of lipid binding sites are compared with sites observed in other TRPs and in Kv channels. These findings suggest PC2, in common with other ion channels, may be modulated by both PIPs and cholesterol, and position PC2 within an emerging model of the roles of lipids in the regulation and organization of ciliary membranes.

Binding site location on GABAA receptors determines whether mixtures of intravenous general anaesthetics interact synergistically or additively in vivo.

General anaesthetics can act on synaptic GABAA receptors by binding to one of three classes of general anaesthetic sites. Canonical drugs that bind selectively to only one class of site are etomidate, alphaxalone, and the mephobarbital derivative, R-mTFD-MPAB. We tested the hypothesis that the general anaesthetic potencies of mixtures of such site-selective agents binding to the same or to different sites would combine additively or synergistically respectively. The potency of general anaesthetics individually or in combinations to cause loss of righting reflexes in tadpoles was determined, and the results were analysed using isobolographic methods. The potencies of combinations of two or three site-selective anaesthetics that all acted on a single class of site were strictly additive, regardless of which single site was involved. Combinations of two or three site-selective anaesthetics that all bound selectively to different sites always interacted synergistically. The strength of the synergy increased with the number of separate sites involved such that the percentage of each agent's EC50 required to cause anaesthesia was just 35% and 14% for two or three sites respectively. Propofol, which binds non-selectively to the etomidate and R-mTFD-MPAB sites, interacted synergistically with each of these agents. The established pharmacology of the three anaesthetic binding sites on synaptic GABAA receptors was sufficient to predict whether a mixture of anaesthetics interacted additively or synergistically to cause loss of righting reflexes in vivo. The principles established here have implications for clinical practice.

Inhibition mechanism of α-glucosidase inhibitors screened from Artemisia selengensis Turcz root

Artemisia selengensis Turcz root (ASTR) is a potential material for screening natural α-glucosidase inhibitors, which effectively reduce postprandial blood glucose level. In this study, natural α-glucosidase inhibitors were screened, and their inhibition mechanism was investigated. Six compounds, including 3,5-dicaffeoylquinic acid (1), 3-caffeyl-5-feruloylquinic acid (3,5-CFQA) (2), 1,3-dicaffeoylquinic acid (3), chlorogenic acid (4), 4-caffeoyl-5-ferulylquinic acid (5) and 3,5-dicaffeoylquinic methyl ester (6), were screened from ASTR through bioactivity-guided isolation. Compounds 2, 5 and 6 were identified from Artemisia selengensis Turcz (AST) for the first time. Given that 3,5-CFQA possessed better inhibitory effect and exhibited considerably lower IC50 value (289.96 μM) than acarbose (600.37 μM), its inhibitory mechanism was investigated. Results showed that 3,5-CFQA reversibly inhibited α-glucosidase activity in a one-way kinetic process through a mixed-type mechanism. The interaction was mostly driven by van der Waals force and hydrogen bonding. The formation of the 3,5-CFQA-α-glucosidase complex involved static quenching and exothermic reaction, which induced the conformational changes in α-glucosidase. Only one class of binding site was found. The results of this work suggested the potential of 3,5-CFQA in inhibiting α-glucosidase activity.

Deciphering essential cistromes using genome-wide CRISPR screens

Although millions of transcription factor binding sites, or cistromes, have been identified across the human genome, defining which of these sites is functional in a given condition remains challenging. Using CRISPR/Cas9 knockout screens and gene essentiality or fitness as the readout, we systematically investigated the essentiality of over 10,000 FOXA1 and CTCF binding sites in breast and prostate cancer cells. We found that essential FOXA1 binding sites act as enhancers to orchestrate the expression of nearby essential genes through the binding of lineage-specific transcription factors. In contrast, CRISPR screens of the CTCF cistrome revealed 2 classes of essential binding sites. The first class of essential CTCF binding sites act like FOXA1 sites as enhancers to regulate the expression of nearby essential genes, while a second class of essential CTCF binding sites was identified at topologically associated domain (TAD) boundaries and display distinct characteristics. Using regression methods trained on our screening data and public epigenetic profiles, we developed a model to predict essential cis-elements with high accuracy. The model for FOXA1 essentiality correctly predicts noncoding variants associated with cancer risk and progression. Taken together, CRISPR screens of cis-regulatory elements can define the essential cistrome of a given factor and can inform the development of predictive models of cistrome function.

ANALYSIS AND DESIGN STRUCTURE OF BUILDING OFFICE AND ADMINISTRATIVE AL-BAHJAH CIREBON

ABSTRACT Al-Bahjah is a formal and non-formal educational institution that combines general education with Al-Quran which is located on PangeranCakrabuana Street,Sumber, Cirebon District. The construction of Building Officeand Administration Al-Bahjah is indispensable as a center for management and administration. The planning of the Building Office and Administrative Al-Bahjah has a floor size of 30 m x 14 m with 5 floors of structure and building height of 19.60 m. The building structure is designed with reference to SNI 1727 - 2013, PPIUG 1987, SNI 2847 - 2013 and SNI 1726 - 2012.The static equivalent analysis method is used in analyzing earthquakes. The structure lies in the class of medium land sites (SD) and is included in the type D Seismic Design Category, so the earthquake force on the structure is planned using a configuration of the structure of the Special Moment Resisting Frame (SMRF).ETABS v9.6 structure analysis program is used to help structure modeling and calculate internal forces acting on the structure, with the material used is concrete f'c 25 Mpa, reinforcementfy 400 Mpa and fy 240 Mpa. Keyword: SNI 1727-2013, PPIUG 1987, SNI 2847-2013, SNI 1726-2013, Seismic Design Category D,Lateral Equivalent Analysis, SMRF, ETABS v9.6.0

Exploiting cationic vacancies for increased energy densities in dual-ion batteries

Dual-ion Li–Mg batteries offer a potential route to cells that combine desirable properties of both single-ion species. To maximize the energy density of a dual-ion battery, we propose a strategy for achieving simultaneous intercalation of both ionic species, by chemically modifying the intercalation host material to produce a second, complementary, class of insertion sites. We show that donor-doping of anatase TiO2 to form large numbers of cationic vacancies allows the complementary insertion of Li+ and Mg2+ in a dual-ion cell with a net increase in cell energy density, due to a combination of an increased reversible capacity, an increased operating voltage, and a reduced polarization. By tuning the lithium concentration in the electrolyte, we achieve full utilization of the Ti4+/Ti3+ redox couple with excellent cyclability and rate capability. We conclude that native interstitial sites preferentially accommodate Li+ ions, while Mg2+ ions occupy single-vacancy sites. We also predict a narrow range of electrochemical conditions where adjacent vacancy pairs preferentially accommodate one ion of each species, i.e., a [LiTi ​+ ​MgTi] configuration. These results demonstrate the implementation of additional host sites such as cationic sites as an effective approach to increase the energy density in dual-ion batteries.

Site index and height-growth curves for Eucalyptus urograndis

Site index curves were adjusted for Eucalyptus urograndis stands located in the municipality of Nova Canaa, MT, using the guide curve method. Six mathematical models were tested to estimate the dominant height of the trees according to age and to verify their accuracy for the definition of site indices. The models tested presented similar values with R² values around 0.40 and errors close to 15%, although none presented expressive superiority in relation to the others. The Schumacher model was used for the construction of curves with four classes of site and with amplitude of three meters each. The model was chosen by the pelasestatisticas presented, for the simplicity and adequate estimation of the heights of the trees with only two parameters, presenting the best graphical distribution of the residues

Evaluating Chromatin Accessibility Differences Across Multiple Primate Species Using a Joint Modeling Approach.

Changes in transcriptional regulation are thought to be a major contributor to the evolution of phenotypic traits, but the contribution of changes in chromatin accessibility to the evolution of gene expression remains almost entirely unknown. To address this important gap in knowledge, we developed a new method to identify DNase I Hypersensitive (DHS) sites with differential chromatin accessibility between species using a joint modeling approach. Our method overcomes several limitations inherent to conventional threshold-based pairwise comparisons that become increasingly apparent as the number of species analyzed rises. Our approach employs a single quantitative test which is more sensitive than existing pairwise methods. To illustrate, we applied our joint approach to DHS sites in fibroblast cells from five primates (human, chimpanzee, gorilla, orangutan, and rhesus macaque). We identified 89,744 DHS sites, of which 41% are identified as differential between species using the joint model compared with 33% using the conventional pairwise approach. The joint model provides a principled approach to distinguishing single from multiple chromatin accessibility changes among species. We found that nondifferential DHS sites are enriched for nucleotide conservation. Differential DHS sites with decreased chromatin accessibility relative to rhesus macaque occur more commonly near transcription start sites (TSS), while those with increased chromatin accessibility occur more commonly distal to TSS. Further, differential DHS sites near TSS are less cell type-specific than more distal regulatory elements. Taken together, these results point to distinct classes of DHS sites, each with distinct characteristics of selection, genomic location, and cell type specificity.

Quantitative glycoproteomics reveals new classes of STT3A- and STT3B-dependent N-glycosylation sites.

Human cells express two oligosaccharyltransferase complexes (STT3A and STT3B) with partially overlapping functions. The STT3A complex interacts directly with the protein translocation channel to mediate cotranslational glycosylation, while the STT3B complex can catalyze posttranslocational glycosylation. We used a quantitative glycoproteomics procedure to compare glycosylation of roughly 1,000 acceptor sites in wild type and mutant cells. Analysis of site occupancy data disclosed several new classes of STT3A-dependent acceptor sites including those with suboptimal flanking sequences and sites located within cysteine-rich protein domains. Acceptor sites located in short loops of multi-spanning membrane proteins represent a new class of STT3B-dependent site. Remarkably, the lumenal ER chaperone GRP94 was hyperglycosylated in STT3A-deficient cells, bearing glycans on five silent sites in addition to the normal glycosylation site. GRP94 was also hyperglycosylated in wild-type cells treated with ER stress inducers including thapsigargin, dithiothreitol, and NGI-1.

Microturbines at Drinking Water Tanks Fed by Gravity Pipelines: A Method and Excel Tool for Maximizing Annual Energy Generation Based on Historical Tank Outflow Data

Wherever the flow of water in a gravity pipeline is regulated by a pressure control valve, hydraulic energy in the form of water pressure can instead be converted into useful mechanical and electrical energy via a turbine. Two classes of potential turbine sites exist—those with (class 1, “buffered”) and those without (class 2, “non-buffered”) a storage tank that decouples inflow from outflow, allowing the inflow regime to be modified to better suit turbine operation. A new method and Excel tool (freely downloadable, at no cost) were developed for determining the optimal hydraulic parameters of a turbine at class 1 sites that maximize annual energy generation. The method assumes a single microturbine with a narrow operating range and determines the optimal design flow rate based on the characteristic site curve and a historical time series of outflow data from the tank, simulating tank operation with a numerical model as it creates a new inflow regime. While no direct alternative methods could be found in the scientific literature or on the internet, three hypothetically applicable methods were gleaned from the German guidelines (published by the German Technical and Scientific Association for Gas and Water (DVGW)) and used as a basis of comparison. The tool and alternative methods were tested for nine sites in Germany.

Integrating Fish Assemblage Data, Modeled Stream Temperatures, and Thermal Tolerance Metrics to Develop Thermal Guilds for Water Temperature Regulation: Wyoming Case Study

AbstractMany streams are experiencing increased average temperatures due to anthropogenic activity and climate change. As a result, surface water temperature regulation is critical for preserving a diverse stream fish species assemblage. The development of temperature regulations has generally been based on laboratory measurements of individual species' thermal tolerances rather than community response to temperature in the field, despite multiple limitations of using laboratory data for this purpose. Using field data to develop temperature regulations may avoid some of the limitations of laboratory data, but the use of field data comes with additional challenges that prevent its widespread adoption. We used Wyoming stream fish assemblages as a case study to examine the feasibility of addressing the limitations of field and laboratory data through a hybrid approach that integrates both types of data to classify species into thermal guilds that can potentially inform regulatory standards. We identified coldwater, coolwater, and warmwater classes of sites with modeled mean August temperatures of <15.5, 15.5–19.9, and >19.9°C, respectively. We used species' associations with these temperature classes to place species into site‐groups. Finally, we used standardized laboratory measures of species' upper acute and chronic thermal tolerances to identify and reclassify species with unusual thermal distributions. Through this process we classified species into five thermal guilds that may be useful for surface water temperature regulation in Wyoming. Our approach addresses the limitations identified for field and laboratory data and demonstrates a framework that could be used for incorporating multiple types of data to develop temperature standards.

Identifying Drugs that Bind Selectively to Intersubunit General Anesthetic Sites in the α1β3γ2 GABAAR Transmembrane Domain.

GABAA receptors (GABAARs) are targets for important classes of clinical agents (e.g., anxiolytics, anticonvulsants, and general anesthetics) that act as positive allosteric modulators (PAMs). Previously, using photoreactive analogs of etomidate ([3H]azietomidate) and mephobarbital [[3H]1-methyl-5-allyl-5-(m-trifluoromethyl-diazirynylphenyl)barbituric acid ([3H]R-mTFD-MPAB)], we identified two homologous but pharmacologically distinct classes of general anesthetic binding sites in the α1β3γ2 GABAAR transmembrane domain at β +-α - (β + sites) and α +-β -/γ +-β - (β - sites) subunit interfaces. We now use competition photolabeling with [3H]azietomidate and [3H]R-mTFD-MPAB to identify para-substituted propofol analogs and other drugs that bind selectively to intersubunit anesthetic sites. Propofol and 4-chloro-propofol bind with 5-fold selectivity to β +, while derivatives with bulkier lipophilic substitutions [4-(tert-butyl)-propofol and 4-(hydroxyl(phenyl)methyl)-propofol] bind with ∼10-fold higher affinity to β - sites. Similar to R-mTFD-MPAB and propofol, these drugs bind in the presence of GABA with similar affinity to the α +-β - and γ +-β - sites. However, we discovered four compounds that bind with different affinities to the two β - interface sites. Two of these bind with higher affinity to one of the β - sites than to the β + sites. We deduce that 4-benzoyl-propofol binds with >100-fold higher affinity to the γ +-β - site than to the α +-β - or β +-α - sites, whereas loreclezole, an anticonvulsant, binds with 5- and 100-fold higher affinity to the α +-β - site than to the β + and γ +-β - sites. These studies provide a first identification of PAMs that bind selectively to a single intersubunit site in the GABAAR transmembrane domain, a property that may facilitate the development of subtype selective GABAAR PAMs.

Distinct G protein-coupled receptor phosphorylation motifs modulate arrestin affinity and activation and global conformation

Cellular functions of arrestins are determined in part by the pattern of phosphorylation on the G protein-coupled receptors (GPCRs) to which arrestins bind. Despite high-resolution structural data of arrestins bound to phosphorylated receptor C-termini, the functional role of each phosphorylation site remains obscure. Here, we employ a library of synthetic phosphopeptide analogues of the GPCR rhodopsin C-terminus and determine the ability of these peptides to bind and activate arrestins using a variety of biochemical and biophysical methods. We further characterize how these peptides modulate the conformation of arrestin-1 by nuclear magnetic resonance (NMR). Our results indicate different functional classes of phosphorylation sites: ‘key sites’ required for arrestin binding and activation, an ‘inhibitory site’ that abrogates arrestin binding, and ‘modulator sites’ that influence the global conformation of arrestin. These functional motifs allow a better understanding of how different GPCR phosphorylation patterns might control how arrestin functions in the cell.

Activation mechanism of ATP-sensitive K+ channels explored with real-time nucleotide binding.

The response of ATP-sensitive K+ channels (KATP) to cellular metabolism is coordinated by three classes of nucleotide binding site (NBS). We used a novel approach involving labeling of intact channels in a native, membrane environment with a non-canonical fluorescent amino acid and measurement (using FRET with fluorescent nucleotides) of steady-state and time-resolved nucleotide binding to dissect the role of NBS2 of the accessory SUR1 subunit of KATP in channel gating. Binding to NBS2 was Mg2+-independent, but Mg2+ was required to trigger a conformational change in SUR1. Mutation of a lysine (K1384A) in NBS2 that coordinates bound nucleotides increased the EC50 for trinitrophenyl-ADP binding to NBS2, but only in the presence of Mg2+, indicating that this mutation disrupts the ligand-induced conformational change. Comparison of nucleotide-binding with ionic currents suggests a model in which each nucleotide binding event to NBS2 of SUR1 is independent and promotes KATP activation by the same amount.

Nucleotide Modulation of KATP Channels Disentangled with FRET

ATP-sensitive K+ channel (KATP) gating is modulated via adenine nucleotide (ATP or ADP) binding to three classes of nucleotide binding site (NBS). The first site is located on the pore-forming Kir subunit of KATP (Kir6.2 in pancreatic β-cells); binding to this NBS is inhibitory. Two stimulatory NBSs are located on the cytoplasmic nucleotide binding domains of the accessory sulfonylurea receptor subunit (SUR1 in β-cells), part of the ABC transporter family. Despite decades of investigation, the details of nucleotide modulation, the affinity and specificity for each NBS, the interactions between the various NBSs, and the mechanism whereby nucleotide binding events are transmitted to the channel pore are largely unknown. To fully understand the nucleotide effects on KATP, we have labelled each NBS (one at a time) with the fluorescent, non-canonical amino acid ANAP and measured nucleotide binding at each site using FRET between ANAP and trinitrophenyl (TNP) nucleotide derivatives in unroofed membrane fragments. This has allowed us to dissect nucleotide binding to each site, evaluate mechanistically mutations expected to affect nucleotide binding (e.g. SUR1-K1384A, Kir6.2-G334D) or channel gating (e.g. Kir6.2-C166S), and investigate the action of clinically important drugs that inhibit or potentiate KATP (sulfonylureas and K+ channel openers). We have also combined this method with patch-clamp electrophysiology (either in separate experiments or simultaneously with patch-clamp fluorometry) to determine the functional consequences of binding at each NBS. This method will not only yield insights into KATP channel activity, but is readily extended to other channels, ABC transporters, and virtually any protein with a suitable fluorescent ligand.

Toward Deep Learning Approaches for Learning Structure Motifs and Classifying Biological Sequences From RNA A-to-I Editing Events

RNA editing is a post-transcriptional RNA sequence modification that alters the mature RNA sequence from its template DNA sequences. RNA editing events are critical in various biological and biochemical mechanisms, and can expand the transcriptomic and proteomic diversity from altered gene regulation to mutations. A-to-I RNA editing is now being vastly detected and quantified on a global scale and gained much attention. A deeper understanding of this process with insufficient genomic annotations and prior knowledge-based filtering steps, such as high-throughput next-generation sequencing techniques, are needed, in addition to data regarding whether an editing location is located in one of the following three main classes of RNA editing sites: ALU in Alu repetitive elements, REP in non-Alu repetitive elements, and NONREP in non-repetitive regions. This study proposes deep learning approaches to ameliorate these issues by learning motif patterns and identifying regions of A-to-I editing events in biological sequences. Using datasets derived from the public REDI portal, 300,000 editing sites were equally divided and used for learning sequence and structure motifs visualized by convolutional kernels. We explored the RNA editing pattern changes using information of positional and class enrichment of learned motifs in the three aforementioned classes. We demonstrated that these newly investigated approaches using large-scale RNA sequencing data offer excellent classification accuracy with a well-optimized convolutional neural network and recurrent neural network classifiers that obtained average area under curves of 0.960 and 0.962, respectively. The findings further decipher the principles underlying RNA editing events and will facilitate more effective RNA sequencing research.

Local genomic features predict the distinct and overlapping binding patterns of the bHLH-Zip family oncoproteins MITF and MYC-MAX.

MITF and MYC are well-known oncoproteins and members of the basic helix-loop-helix leucine zipper (bHLH-Zip) family of transcription factors (TFs) recognizing hexamer E-box motifs. MITF and MYC not only share the core binding motif, but are also the two most highly expressed bHLH-Zip transcription factors in melanocytes, raising the possibility that they may compete for the same binding sites in select oncogenic targets. Mechanisms determining the distinct and potentially overlapping binding modes of these critical oncoproteins remain uncharacterized. We introduce computational predictive models using local sequence features, including a boosted convolutional decision tree framework, to distinguish MITF versus MYC-MAX binding sites with up to 80% accuracy genomewide. Select E-box locations that can be bound by both MITF and MYC-MAX form a separate class of MITF binding sites characterized by differential sequence content in the flanking region, diminished interaction with SOX10, higher evolutionary conservation, and less tissue-specific chromatin organization.

HIV-1 Protease Uses Bi-Specific S2/S2′ Subsites to Optimize Cleavage of Two Classes of Target Sites

Retroviral proteases (PRs) have a unique specificity that allows cleavage of sites with or without a P1′ proline. A P1′ proline is required at the MA/CA cleavage site due to its role in a post-cleavage conformational change in the capsid protein. However, the HIV-1 PR prefers to have large hydrophobic amino acids flanking the scissile bond, suggesting that PR recognizes two different classes of substrate sequences. We analyzed the cleavage rate of over 150 combinations of six different HIV-1 cleavage sites to explore rate determinants of cleavage. We found that cleavage rates are strongly influenced by the two amino acids flanking the amino acids at the scissile bond (P2–P1/P1′–P2′), with two complementary sets of rules. When P1′ is proline, the P2 side chain interacts with a polar region in the S2 subsite of the PR, while the P2′ amino acid interacts with a hydrophobic region of the S2′ subsite. When P1′ is not proline, the orientations of the P2 and P2′ side chains with respect to the scissile bond are reversed; P2 residues interact with a hydrophobic face of the S2 subsite, while the P2′ amino acid usually engages hydrophilic amino acids in the S2′ subsite. These results reveal that the HIV-1 PR has evolved bi-functional S2 and S2′ subsites to accommodate the steric effects imposed by a P1′ proline on the orientation of P2 and P2′ substrate side chains. These results also suggest a new strategy for inhibitor design to engage the multiple specificities in these subsites.